Methods and systems for detecting an analyte or classifying a sample

Abstract

The present invention relates to methods and systems for detecting one or more analytes in a sample and/or for classifying a sample. In particular, the present invention relates to methods and systems which can be used to detect the analytes in real time and which rely on flowing through a microfluidic device one or more types of sensor molecule each comprising a domain that binds one or more analytes, a chemiluminescent donor domain and an acceptor domain, wherein the separation and relative orientation of the chemiluminescent donor domain and the acceptor domain, in the presence and/or the absence of analyte, is within +50% of the Forster distance.

The invention claimed is: 
     
       1. A method of detecting an analyte in a sample, the method comprising
 i) flowing through a microfluidic device comprising one or more microchannels,
 a) the sample, 
 b) a sensor molecule comprising a domain that binds the analyte, a chemiluminescent donor domain and an acceptor domain, wherein the separation and relative orientation of the chemiluminescent donor domain and the acceptor domain, in the presence and/or the absence of analyte, is within ±50% of the Forster distance, and wherein the sensor molecule is not fixed to the device, 
 c) a substrate of the chemiluminescent donor, 
 
 ii) mixing the sensor molecule, sample and substrate in the device, and 
 iii) detecting modification of the substrate by the chemiluminescent donor using an electro-optical sensing device and calculating as a ratio the energy transfer occurring between the chemiluminescent donor domain and the acceptor domain, 
 wherein the spatial location and/or dipole orientation of the chemiluminescent donor domain relative to the acceptor domain is altered when the analyte binds the sensor molecule. 
 
     
     
       2. The method of  claim 1 , wherein the method can be used to detect the analyte in real time and/or which is performed within about 1 s to about 100 s. 
     
     
       3. The method of  claim 1 , wherein the sensor molecule and substrate enter the device through different microchannels. 
     
     
       4. The method of  claim 1 , wherein one or more or all of the following features apply;
 i) the Forster distance of the chemiluminescent donor domain and the acceptor domain is at least 5.6 nm, 
 ii) the analyte binding or releasing from the sensor molecule results in a change in BRET ratio which is ≥15% of the maximum observed BRET ratio, or 
 iii) the acceptor domain has a Stokes Shift of between about 50 nm and about 150 nm. 
 
     
     
       5. The method of  claim 1 , wherein the quantum yield detected by the electro-optical sensing device is less than about 8%, or less than about 5%, or less than about 2%. 
     
     
       6. The method of  claim 1 , wherein the sample is a liquid, gas, liquid which has been pre-equilibrated with a gas, emulsion or suspension. 
     
     
       7. The method of  claim 1 , wherein
 i) the flow rate through the microfluidic device is between about 1 μl/hour to about 1.5 ml/hour, and/or 
 ii) flowing through the microfluidic device is continuous flow, batch flow or stop flow. 
 
     
     
       8. The method of  claim 1 , wherein step iii) is performed in a reaction chamber with a volume of about 1 pl to about 200 μl. 
     
     
       9. The method of  claim 1 , wherein the domain that binds the analyte is a protein or a nucleic acid. 
     
     
       10. The method of  claim 9 , wherein the protein is a receptor, odorant binding protein, pheromone-binding protein, enzyme, ligand carrier, bacterial periplasmic binding protein or a G protein coupled receptor. 
     
     
       11. The method of  claim 1 , wherein one or more or all of the following features apply;
 i) the chemiluminescent donor domain is a bioluminescent protein, 
 ii) the substrate is luciferin, calcium, coelenterazine, or a derivative or analogue of coelenterazine, or, 
 ii) the acceptor domain is a fluorescent acceptor domain. 
 
     
     
       12. The method of  claim 1 , wherein
 i) the bioluminescent protein is a luciferase or a biologically active variant or fragment, and/or 
 ii) the substrate is luciferin, coelenterazine, or a derivative or analogue of coelenterazine, and/or 
 iii) the acceptor domain is green fluorescent protein (GFP), Venus, mOrange, or a biologically active variant or fragment of any one thereof. 
 
     
     
       13. The method of  claim 1  which comprises simultaneously or sequentially detecting two or more different analytes using the same microfluidic device. 
     
     
       14. The method of  claim 1 , wherein the microfluidic device comprises one or more sets of
 a) three input microchannels, one each for the sensor molecule, substrate and sample, or 
 b) two input microchannels, one for the substrate and the other for a pre-mixture of the sensor molecule and sample, or 
 c) two input microchannels, one for the sensor molecule and the other for a pre-mixture of the substrate and sample. 
 
     
     
       15. The method of  claim 1 , wherein one or more or all of the following features apply:
 i) at least one microchannel comprises a reaction chamber which has a different volume to at least one other microchannel; 
 ii) at least one microchannel comprises two or more reaction chambers of the same or different volume, or 
 iii) the electro-optical sensing device has at least two different wavelength channels. 
 
     
     
       16. A microfluidic system comprising
 i) at least one reservoir suitable for containing a sensor molecule, 
 ii) a sensor molecule comprising a domain that binds the analyte, a chemiluminescent donor domain and an acceptor domain, wherein the separation and relative orientation of the chemiluminescent donor domain and the acceptor domain, in the presence and/or the absence of analyte, is within ±50% of the Forster distance, 
 iii) a microfluidic device comprising one or more microchannels, 
 iv) means for mixing the sensor molecule, the sample and a substrate of the chemiluminescent donor domain in the device, 
 v) a reaction chamber for detecting binding of the analyte to the sensor molecule, and 
 vi) an electro-optical sensing device, 
 wherein the spatial location and/or dipole orientation of the chemiluminescent donor domain relative to the acceptor domain is altered when the analyte binds the sensor molecule, and 
 wherein the sensor molecule is not fixed to the microfluidic device. 
 
     
     
       17. The system of  claim 16 
 i) which can be used to detect the analyte in real time, and/or 
 ii) wherein the microfluidic device is designed to enable the detection of two or more analytes. 
 
     
     
       18. A method of classifying a sample, the method comprising
 i) flowing through a microfluidic device comprising one or more microchannels,
 a) the sample, 
 b) a sensor molecule comprising a domain that binds one or more analytes, a chemiluminescent donor domain and an acceptor domain, wherein the separation and relative orientation of the chemiluminescent donor domain and the acceptor domain, in the presence and/or the absence of analyte(s), is within ±50% of the Forster distance, and wherein the sensor molecule is not fixed to the device, 
 c) a substrate of the chemiluminescent donor, 
 
 ii) mixing the sensor molecule, sample and substrate in the device, 
 iii) detecting modification of the substrate by the chemiluminescent donor using an electro-optical sensing device and calculating as a ratio the energy transfer occurring between the chemiluminescent donor domain and the acceptor domain, 
 iv) processing at least one signal from the electro-optical sensing device and correlating the pattern of electro-optical responses with one or more pre-determined characteristics of one or more samples of interest, and 
 v) classifying the sample based on the correlation of the pattern of responses, 
 wherein the spatial location and/or dipole orientation of the chemiluminescent donor domain relative to the acceptor domain is altered when the one or more analytes binds the sensor molecule. 
 
     
     
       19. The method of  claim 18 , wherein one or more of the analytes are unknown.